基因治疗用质粒DNA的制备工艺

Development of A Procedure for Preparation of Plasmid DNA for Gene Therapy

目的探索基因治疗用质粒DNA的制备工艺。方法以CaCl2沉淀法去除大分子RNA,Q-Sepharose和SOURCE两步离子交换层析法去除染色体DNA、小分子RNA、蛋白质等杂质,并对各步骤样品进行检测。结果质粒pIRVP3HNIL18终产品的产量为1.638mg质粒DNA/g细菌,纯度为1.839,相对回收率为12.55%,蛋白质含量为0.0028mg/ml,未检测到RNA和染色体DNA。结论此工艺可有效去除质粒DNA制备过程中产生的杂质,可应用于药剂水平质粒DNA的制备。

Objective To explore a procedure for preparation of plasmid DNA for gene therapy. Methods Remove macromolecular RNA from cultured recombinant E. coli with plasmid pIRVP3HNIL18 by calcium chloride precipitation, and other impurities such chromosomal DNA, micromolecular RNA and protein by Q-Sepharose and SOURCE ion exchange chromatography. Results The yield, purity, relative recovery and protein content of final product of plasmid pIRVP3HNIL18 were 1. 638 mg plasmid DNA/g bacteria, 1. 839,12. 55% and 0. 002 8 mg/ml respectively. No RNA or chromosomal DNA was detected. Conclusion The developed procedure was effective for removal of impurities and suitable for preparation of therapeutic-grade plasmid DNA.